This staining was performed to detect fetuin-A in astrocytes. Fetuin-A and astrocytes were detected in paraffin sections (1 \u03bcm thickness) of formalin-fixed human brain tissue. Glial Fibrillary Acid Antigen (GFAP) was marked by a polyclonal rabbit-anti-human antibody (Spring Bioscience Cat# E18320, RRID:AB_1661177, dilution 1:50) and a polyclonal goat-anti-rabbit Alexa Fluor\u00ae 488 conjugated secondary antibody (Thermo Fisher Scientific Cat# A-11070, RRID:AB_2534114, dilution 1:300).Fetuin-A was detected by using a monoclonal IgG2a mouse-anti-human antibody (clone MAHS-1, dilution 1.0 \u00b5g\/mL), raised against purified human fetuin-A in our laboratories. Antibody binding was detected by tyramide signal amplification using a secondary biotinylated polyclonal goat-anti-mouse antibody (Dako Cat# E0433, RRID:AB_2687905, dilution 1:300) and a Tyramide Signal Amplification Kit (Life Technologies, Carlsbad, USA, T-20933). To minimize lipofuscin autofluorescence, sections were counterstained with Sudan Black (Sigma-Aldrich, Munich, Germany, 199664, dilution 0.3% in 70% ethanol, 5 minutes). Nuclei were stained with DAPI (Sigma-Aldrich, Munich, Germany D9542, dilution 0.25 \u00b5g\/ml, 5 minutes). Sections were mounted with Immumount (Thermo Scientific, Waltham, USA, 9990402) and stored at 8\u00b0C in the dark.